Complete Inhibition Of Protein Synthesis Research Articles

The role of the encephalomyocarditis virus type 1 proteins L and 2A in the inhibition of the synthesis of cellular proteins and the accumulation of viral proteins during infection

Infection of cells with encephalomyocarditis virus type 1 (EMCV-1, Cardiovirus A: Picornaviridae) is accompanied by suppression of cellular protein synthesis. The main role in the inhibition of cellular translation is assigned to the L and 2A «security» proteins. The mechanism of the possible influence of the L protein on cellular translation is unknown. There are hypotheses about the mechanism of influence of 2A protein on the efficiency of cap-dependent translation, which are based on interaction with translation factors and ribosome subunits. However, the available experimental data are contradictory, obtained using different approaches, and do not form a unified model of the interaction between the L and 2A proteins and the cellular translation machinery. To study the role of L and 2A «security» proteins in the suppression of translation of cellular proteins and the efficiency of translation and processing of viral proteins in infected cells. Mutant variants of EMCV-1 were obtained to study the properties of L and 2A viral proteins: Zfmut, which has a defective L; Δ2A encoding a partially deleted 2A; Zfmut&Δ2A containing mutations in both proteins. Translational processes in infected cells were studied by Western-blot and the pulse method of incorporating radioactively labeled amino acids (14C) into newly synthesized proteins, followed by radioautography. The functional inactivation of the 2A protein does not affect the inhibition of cellular protein synthesis. A direct correlation was found between the presence of active L protein and specific inactivation of cellular protein synthesis at an early stage of viral infection. Nonspecific suppression of the translational processes of the infected cell, accompanied by phosphorylation of eIF2α, occurs at the late stage of infection. Partial removal of the 2A protein from the EMCV-1 genome does not affect the development of this process, while inactivation of the L protein accelerates the onset of complete inhibition of protein synthesis. Partial deletion of the 2A disrupts the processing of viral capsid proteins. Suppression of L protein functions leads to a decrease in the efficiency of viral translation. A study of the role of EMCV-1 L and 2A proteins during the translational processes of an infected cell, first performed using infectious viral pathogens lacking active L and 2A proteins in one experiment, showed that 2A protein is not implicated in the inhibition of cellular translation in HeLa cells; L protein seems to play an important role not only in the specific inhibition of cellular translation but also in maintaining the efficient synthesis of viral proteins; 2A protein is involved not only in primary but also in secondary processing of EMCV-1 capsid proteins.

OR16-1 How Strong and Sustained Activation of the Estrogen Receptor-mediated Anticipatory Unfolded Protein Response Kills Breast and Ovarian Cancer Cells

Abstract In orthotopic xenografts and a PDX, our anticancer drug ErSO eradicates primary and metastatic therapy-resistant estrogen receptor alpha (ERα) positive breast cancer, and induces near-complete regression of ovarian cancer. The mechanism by which ErSO induces necrosis and kills ER positive cancer cells was unknown. From genome-wide CRISPR-Cas9 screens in MCF-7 and T47D cells with negative selection against first-generation BHPI and second-generation ErSO, and follow-on experiments, we identified the Ca2+ activated, plasma membrane Na+ channel TRPM4 as the executioner protein that BHPI and ErSO use to induce necrosis. Notably, in 6 ERα+ breast and ovarian cancer cell lines, knockout of TRPM4 completely abolished the ability of ErSO to induce death of cancer cells. Moreover, TRPM4 mRNA and protein were dramatically down-regulated in breast cancer cells selected for resistance to BHPI and ErSO. Furthermore, in a mouse xenograft, while ErSO induced near complete regression of orthotopic MCF-7-ERY537S-luciferase tumors, ErSO had no effect on the TRPM4 knockout tumors, which continued their robust growth. Since necrosis, but not most other death pathways, activates immune cells, inducing immunogenic cell death, this provides a new avenue for enhancing cancer immunotherapy. Importantly, medium from ErSO-treated wild type MCF-7 cells, but not medium from TRPM4 knockout cells, robustly activates human THP-1 monocytes and greatly increases their migration. BHPI and ErSO-induced initial anticipatory unfolded protein response (a-UPR) activation results in elevated cytosolic Ca2+, opening the plasma membrane TRPM4 channel, eliciting a rapid influx of external Na+, accompanying Cl- to balance the charge, and water to maintain osmolality. This swells the cells, causing osmotic stress. Importantly, it is the osmotic stress that sustains UPR hyperactivation, leading to ATP depletion, which contributes to membrane rupture and rapid necrotic cell death and to near complete inhibition of protein synthesis that ultimately kills any surviving cancer cells . Suggesting a broad role of TRPM4 in the actions of necrosis inducing anticancer drugs, TRPM4 knockout also inhibited necrosis induced by unrelated anticancer therapies, the mitochondrial targeting oncolytic peptide, LTX-315 and the Ca2+ channel targeting agent, Englerin A. Since increasing expression TRPM4 by viral transduction results in progressively increased sensitivity of ER positive breast cancer cells to killing by ErSO, this enables identification of breast cancer patients whose elevated TRPM4 levels make them most likely to benefit from this novel therapy. The TRPM4 pathway is a new mechanism for sustained lethal activation of the UPR and for targeting ER positive breast and ovarian cancer. Presentation: Sunday, June 12, 2022 11:00 a.m. - 11:15 p.m.

Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

Ricin, Shiga toxin, exotoxin A, and diphtheria toxin are AB-type protein toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. Intoxication is therefore viewed as an irreversible process. Using flow cytometry and a fluorescent reporter system to monitor protein synthesis, we show a single molecule of cytosolic toxin is not sufficient for complete inhibition of protein synthesis or cell death. Furthermore, cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin challenge. Thus, in contrast to the prevailing model, ongoing toxin delivery to the cytosol appears to be required for the death of cells exposed to sub-optimal toxin concentrations.

Combination treatments with ABT‐263 and an immunotoxin produce synergistic killing of ABT‐263‐resistant small cell lung cancer cell lines

Synergistic killing was achieved when Small Cell Lung Cancer (SCLC) cell lines were incubated with ABT-263 and an immunotoxin directed to the transferrin receptor. SCLC lines are variably sensitive to the BH-3 only peptide mimetic, ABT-263. To determine their sensitivity to toxin-based reagents, we incubated four representative SCLC lines with a model Pseudomonas exotoxin-based immunotoxin directed to the transferrin receptor. Remarkably in 4-of-4 lines, there was little evidence of immunotoxin-mediated cytotoxicity despite near complete inhibition of protein synthesis. However, when combinations of ABT-263 and immunotoxin were added to the ABT-263-resistant cell lines (H196 and H69AR), there was synergistic killing as evidenced by increased activation of caspase 3/7, annexin V staining, and loss of cell integrity. Synergistic killing was evident at 6 hr and correlated with loss of Mcl-1. This synergy was also noted when the closely related compound ABT-737 was combined with the same immunotoxin. To establish that the synergy seen in tissue culture could be achieved in vivo, H69AR cells were grown as tumors in nude mice and shown to be susceptible to the killing action of an immunotoxin-ABT-737 combination but not to either agent alone. When immunotoxin-ABT combinations were added to ABT-263-sensitive lines (H146 and H1417), killing was additive. Our data support combination approaches for treating ABT-263-resistant SCLC with ABT-263 and a second agent that provides synergistic killing action.

Abstract B44: Combination treatments of SCLC with ABT-263 and Immunotoxins produces synergistic killing of ABT-263-resistant cells

Abstract Small cell lung cancer (SCLC) cell lines are variably sensitive to the BH-3 only peptide mimetic, ABT-263. To determine their sensitivity to toxin-based reagents, we incubated four representative SCLC lines with a model Pseudomonas exotoxin-based immunotoxin directed to the transferrin receptor. Remarkably in 4-of-4 lines, there was little evidence of immunotoxin-mediated cytotoxicity despite near complete inhibition of protein synthesis. However, when combinations of ABT-263 and immunotoxin were added to the ABT-263-resistant cell lines (H196 and H69AR), there was synergistic killing as evidenced by increased activation of caspase 3/7, annexin V staining and loss of cell integrity. This synergy was also noted when the closely related compound ABT-737 was combined with the same immunotoxin. To establish that the synergy seen in tissue culture could also be achieved in vivo, H69AR cells were grown as tumors in nude mice and shown to be susceptible to the killing action of an immunotoxin-ABT combination but not to either agent alone. When immunotoxin-ABT combinations were added to ABT-263-sensitive lines (H146 and H889), killing was primarily due to the action of ABT-263. Our data support combination approaches for treating ABT-263-resistant SCLC with ABT-263 and a second agent that provides synergistic killing action.

ABT-737 Overcomes Resistance to Immunotoxin-Mediated Apoptosis and Enhances the Delivery of Pseudomonas Exotoxin–Based Proteins to the Cell Cytosol

Pseudomonas exotoxin (PE)-based immunotoxins (antibody-toxin fusion proteins) have achieved frequent complete remissions in patients with hairy cell leukemia but far fewer objective responses in other cancers. To address possible mechanisms of resistance, we investigated immunotoxin activity in a model system using the colon cancer cell line, DLD1. Despite causing complete inhibition of protein synthesis, there was no evidence that an immunotoxin targeted to the transferrin receptor caused apoptosis in these cells. To address a possible protective role of prosurvival Bcl-2 proteins, the BH3-only mimetic, ABT-737, was tested alone or in combination with immunotoxins. Neither the immunotoxin nor ABT-737 alone activated caspase 3, whereas the combination exhibited substantial activation. In other epithelial cell lines, ABT-737 enhanced the cytotoxicity of PE-related immunotoxins by as much as 20-fold, but did not enhance diphtheria toxin or cycloheximide. Because PE translocates to the cytosol via the endoplasmic reticulum (ER) and the other toxins do not, ABT-737-mediated effects on the ER were investigated. ABT-737 treatment stimulated increased levels of ER stress response factor, ATF4. Because of its activity in the ER, ABT-737 might be particularly well suited for enhancing the activity of immunotoxins that translocate from the ER to the cell cytosol.

NF-kappaB is required for apoptosis prevention during herpes simplex virus type 1 infection.

Wild-type herpes simplex virus type 1 (HSV-1) infection triggers apoptosis in human cells. The subsequent synthesis of infected cell proteins between 3 and 6 h postinfection (hpi) acts to block this process from killing the cells. The factors produced during this window also prevent cell death induced by environmental staurosporine or sorbitol (M. Aubert, J. O'Toole, and J. A. Blaho, J. Virol. 73:10359-10370, 1999). We now report that (i) during the prevention window, HSV-1(F) also inhibited apoptosis induced by tumor necrosis factor alpha (TNF-alpha) plus cycloheximide (CHX) treatment. While deciphering the mechanism of this inhibition, we observed that (ii) the transcription factor NF-kappaB translocated from the cytoplasm into the nuclei of infected cells, and (iii) this migration initiated at 3 hpi. (iv) The complete inhibition of protein synthesis at 3 hpi by the addition of CHX precluded NF-kappaB translocation, while CHX additions at 6 hpi or later did not elicit this effect. This result confirms that infected cell protein synthesis is required for the nuclear import of NF-kappaB. (v) The detection of NF-kappaB in nuclei correlated with the ability of HSV-1(F), HSV-1(KOS1.1), or HSV-1(R7032), a replication-competent recombinant virus containing a deletion in the gene encoding the gE glycoprotein, to prevent apoptosis. (vi) NF-kappaB did not bind its kappaB DNA recognition site and remained cytoplasmic in cells actively undergoing apoptosis following infection with HSV-1(vBSdelta27), a virus with the key regulatory protein ICP27 deleted. (vii) Prestimulation of NF-kappaB by the addition of a phorbol ester prevented HSV-1(vBSdelta27)-induced apoptosis. (viii) Retention of NF-kappaB in the cytoplasm by the addition of a pharmacological antagonist of its release from IkappaBalpha led to an increase in death factor processing during HSV-1(F) infection. (ix) A novel HEp-2 clonal cell line, termed IkappaBalphaDN, was generated which expresses a dominant-negative form of IkappaBalpha. Treatment of IkappaBalphaDN cells with TNF-alpha in the absence of CHX resulted in apoptotic death due to the inability of NF-kappaB to become activated in these cells. Finally, (x) infection of IkappaBalphaDN cells with HSV-1(F) or HSV-1(KOS1.1) resulted in apoptosis, demonstrating that (xi) the nuclear translocation of NF-kappaB between 3 and 6 hpi (the prevention window) is necessary to prevent apoptosis in wild-type HSV-1-infected human HEp-2 cells.

Ribonuclease Activity of Rat Liver Perchloric Acid-soluble Protein, a Potent Inhibitor of Protein Synthesis

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 microM led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [alpha-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule.

Nascent Flavivirus RNA Colocalizedin Situwith Double-Stranded RNA in Stable Replication Complexes

Incorporation of bromouridine (BrU) into viral RNA in Kunjin virus-infected Vero cells treated with actinomycin D was monitoredin situby immunofluorescence using antibodies reactive with Br-RNA. The results showed unequivocally that nascent viral RNA was located focally in the same subcellular site as dsRNA, the putative template for flavivirus RNA synthesis. When cells were labeled with BrU for 15 min, the estimated cycle period for RNA synthesis, the nascent Br-RNA was not digested in permeabilized cells by RNase A under high-salt conditions, in accord with our original model of flavivirus RNA synthesis (Chu, P. W. G., and Westaway, E. G.,Virology140, 68–79, 1985). The model assumes that there is on average only one nascent strand per template, which remains bound until displaced during the next cycle of RNA synthesis. The replicase complex located by BrU incorporation in the identified foci was stable, remaining active in incorporating BrU or [32P]orthophosphate in viral RNA after complete inhibition of protein synthesis in cycloheximide-treated cells. These results are in accord with our proposal that dsRNA detected in foci previously located by immunofluorescence or by immunogold labeling of induced vesicle packets is functioning as the true replicative intermediate (Westawayet al., J. Virol.71, 6650–6661, 1997; Mackenzieet al., Virology245, 203–215, 1998). Implications are that the replicase complex is able to recycle in the same membrane site in the absence of continuing protein synthesis and that possibly apart from uncleaved NS3–NS4A, it has no requirement for a polyprotein precursor late in infection.

Depletion of neuronal endoplasmic reticulum calcium stores by thapsigargin: effect on protein synthesis.

We have used thapsigargin (TG), a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca(2+)-ATPases, and caffeine, an agonist of the ryanodine receptor, to study the effect of emptying of ER calcium stores on protein synthesis in neuronal cells. TG at 1 microM caused a permanent inhibition of protein synthesis in hippocampal slices from 3-week-old rats but no inhibition in slices prepared from 2-month-old animals. Caffeine at 10 mM caused a reduction of protein synthesis in both 3-week- and 2-month-old rats immediately after exposure, but complete recovery of protein synthesis occurred within 30 min after treatment. In neuronal cells, TG produced an almost complete inhibition of protein synthesis that was only partially reversed over a 24-h recovery period. TG did not significantly affect neuronal ATP levels or energy charge. Fifty percent inhibition of protein synthesis was achieved with approximately 5 nM TG. Recovery of protein synthesis after TG treatment was significantly hindered when serum was omitted from the medium after TG exposure, suggesting that serum promotes recovery of ER calcium homeostasis. It is concluded that TG is a suitable tool for the study of the mechanisms of protein synthesis inhibition after transient cerebral ischemia. The possibility that disturbances in ER calcium homeostasis may contribute to the pathological process of ischemic cell death is discussed.

Tetrahydroaminoacridine-induced apoptosis in rat hepatocytes

Tacrine (tetrahydroaminoacridine, THA) is currently administered to thousands of patients for the treatment of Alzheimer's disease. Unfortunately, THA therapy is often limited by this drugs' propensity to induce reversible hepatotoxicity. In the present study we investigated the mechanism of THA cytotoxicity by measuring the effect of THA on cell viability, protein synthesis activity and the induction of apoptosis in suspensions of freshly isolated rat hepatocytes. Our experimental findings indicate that THA-mediated apoptosis is responsible for the acute in vitro hepatotoxicity observed with this aminoacridine derivative. We found that THA-treated hepatocytes (0.1, 0.25 and 0.5 m m) demonstrated a significant and dose-dependent reduction in cellular protein synthesis activity (84, 55 and 5% of control activity, respectively) after 1 hr of incubation. However, in hepatocytes exposed to 0.1 and 0.25 m m THA, the inhibition of protein synthesis was short-lived. In these treated cells, protein synthesis activity returned to control levels (100%) by the fifth hr of incubation without a significant increase in cellular lactate dehydrogenase (LDH) leakage or the induction of apoptosis. In hepatocytes exposed to 0.5 m m THA, the near complete inhibition of protein synthesis was not reversible and a dramatic increase in LDH leakage (necrosis) was observed after 6 hr of treatment. In 0.5 m m THA-treated hepatocytes the appearance of apoptotic nuclei and cells were observed with electron microscopy following 2 hr of treatment (12% of total hepatocytes analysed) and steadily increased to 42% by the fifth hr (compared with 4% for control cells). We speculate that THA's ability to inhibit hepatocyte protein synthesis (>50%) and induce apoptosis may have an important role in the hepatotoxic episodes experienced by Alzheimer's patients taking this drug. However, the role of apoptosis in clinical THA-induced hepatotoxicity and the relevance of using rat hepatocyte suspensions as an in vitro model for THA hepatotoxicty in vivo require additional investigation.

Expression of urokinase-type plasminogen activator, its receptor and type-1 plasminogen activator inhibitor is differently regulated by inhibitors of protein synthesis in human cancer cell lines

Expression of the various components of the plasminogen activation system is under tight regulation by hormones, cytokines, and growth factors under physiologic conditions. Like early-response genes, these components are modulated by inhibitors of protein synthesis in some cell lines. To clarify the specific expression and regulation of mRNAs for urokinase (uPA), its receptor (uPAR), and type-1 plasminogen activator inhibitor (PAI-1), I analyzed RNA from four human cancer cell lines by RNA blotting after treatment with cycloheximide, anisomycin, emetine or puromycin. These inhibitors, all of which induced translational arrest, induced a very diverse response in the various transcripts, suggesting that the inhibitors mediate their effects through different molecular mechanisms. Dose-response analysis showed that, in A549 cells, anisomycin strongly induced uPAR and PAI-1 mRNA at concentrations that did not cause complete inhibition of protein synthesis, whereas cycloheximide induced these transcripts in a dose-dependent manner only at concentrations sufficient to inhibit total protein synthesis by >90%. Puromycin induced the 3.4-kb transcript of PAI-1 mRNA in A549 and RD cells, whereas it decreased the expression of both the 3.4-kb and 2.4-kb PAI-1 transcripts in HT-1080 cells. Different time patterns of induction for uPA, uPAR and PAI-1 mRNA suggest that even in the same cell type, inhibitors of protein synthesis mediate their effects on various genes through different mechanisms. Thus, induction of uPA, uPAR and PAI-1 transcripts by inhibitors of protein synthesis was dependent on the gene, the cell line, and the type of inhibitor, and inhibition of protein synthesis per se was not sufficient for induction of these transcripts.

Inhibition of Protein Synthesis by RA-VII

The antitumor natural product RA-VII has been evaluated as an inhibitor of protein synthesis in vitro. Complete inhibition of protein synthesis in rabbit reticulocyte lysates is observed with 5 μM RA-VII. Mechanistic studies using purified elongation factors and ribosomes identify RA-VII as a peptidyltransferase inhibitor. Thus, similar to the related natural products bouvardin and RA-XII, RA-VII appears to function by binding to eukaryotic ribosomes.

Induction of P‐Glycorprotein mRNA by protein synthesis inhibition is not controlled by a transcriptional repressor protein in rat and human liver cells

Recent studies have suggested that a labile transcriptional repressor protein is important in the regulation of pgp mRNA expression. However, cycloheximide (CHX) the protein synthesis inhibitor used, can increase mRNAs by either stabilizing the mRNA transcript or directly activating gene transcription. To determine whether CHX posttranscriptionally increased pgp mRNA, we compared the effect of CHX, which inhibits protein synthesis by stabilizing polysomes, with puromycin (PURO), which inhibits protein synthesis by polysome destabilization. In rat hepatocytes, CHX induced pgp2 mRNA, and the increase was proportional to the degree of protein synthesis inhibition. In contrast, despite almost complete inhibition of protein synthesis, PURO did not induce pgp2 mRNA. Further studies demonstrated that PURO pretreatment could block pgp2 mRNA induction by CHX. Likewise, in cultures of primary human hepatocytes CHX, but not PURO, induced MDR1 mRNA. A polymerase chain reaction assay was developed to assess whether CHX treatment altered the length of the 3'-untranslated region (UTR) of pgp2. CHX treatment time dependently increased the length of the pgp2 3'-UTR. To determine whether CHX acts as a transcriptional agonist, we performed nuclear run-off analysis and found no increase in pgp2 gene transcription compared to untreated control. Further, transcription studies were performed by transiently transfecting HepG2 cells with plasmids containing 5' segments of human MDR1 fused with the reporter chloramphenicol acetyltransferase (CAT). These plasmids were not transcriptionally activated by CHX. In summary, our results cast doubt on the existence of a labile transcriptional repressor protein for pgp. Furthermore, these are the first studies to demonstrate that polysomal destabilization by PURO can block CHX induction of pgp.

Tetrahydroaminoacridine–Induced Ribosomal Changes and Inhibition of Protein Synthesis in Rat Hepatocyte Suspensions

ABSTRACT Tacrine (tetrahydroaminoacridine) is currently the only drug approved for the treatment of Alzheimer's disease. Unfortunately, tetrahydroaminoacridine therapy is often limited by this drug's propensity to induce reversible hepatotoxicity. Using suspensions of freshly isolated rat hepatocytes, we investigated the mechanism of tetrahydroaminoacridine cytotoxicity by examining the effect of tetrahydroaminoacridine on hepatocyte viability, protein synthesis, protein, DNA and RNA levels and ultrastructure. Our experimental findings support the explanation that tetrahydroaminoacridine–induced hepatotoxicity results from tetrahydroaminoacridine's adverse effect on protein synthesis and ribosomal structure and function. We found that viable, tetrahydroaminoacridine–treated hepatocytes (1.0 to 2.0 mmol/L or 118 to 235 μg/106 cells) demonstrated a dose–dependent and dramatic aggregation of ribosomes on endoplasmic reticulum as well as the aggregation of other nucleic acids found in the nucleus (chromatin) and in mitochondria. These electron microscopy data suggest that tetrahydroaminoacridine treatment results in severe ribosomal dysfunction. This was confirmed by the observed rapid loss of cellular RNA content (but not DNA or protein) and the rapid and complete inhibition of protein synthesis in tetrahydroaminoacridine–treated cells (lowest concentration tested was 0.5 mmol/L or 58 μg/106 cells). Thus tetrahydroaminoacridine treatment appears to aggregate hepatocellular nucleic acids, and in doing so adversely affects ribosomal function and protein synthesis. We propose that these adverse effects of exposure to tetrahydroaminoacridine are responsible for tetrahydroaminoacridine–induced hepatotoxicity. (Hepatology 1994;20:•••.)

Tetrahydroaminoacridine-induced ribosomal changes and inhibition of protein synthesis in rat hepatocyte suspensions.

Tacrine (tetrahydroaminoacridine) is currently the only drug approved for the treatment of Alzheimer's disease. Unfortunately, tetrahydroaminoacridine therapy is often limited by this drug's propensity to induce reversible hepatotoxicity. Using suspensions of freshly isolated rat hepatocytes, we investigated the mechanism of tetrahydroaminoacridine cytotoxicity by examining the effect of tetrahydroaminoacridine on hepatocyte viability, protein synthesis, protein, DNA and RNA levels and ultrastructure. Our experimental findings support the explanation that tetrahydroaminoacridine-induced hepatotoxicity results from tetrahydroaminoacridine's adverse effect on protein synthesis and ribosomal structure and function. We found that viable, tetrahydroaminoacridine-treated hepatocytes (1.0 to 2.0 mmol/L or 118 to 235 micrograms/10(6) cells) demonstrated a dose-dependent and dramatic aggregation of ribosomes on endoplasmic reticulum as well as the aggregation of other nucleic acids found in the nucleus (chromatin) and in mitochondria. These electron microscopy data suggest that tetrahydroaminoacridine treatment results in severe ribosomal dysfunction. This was confirmed by the observed rapid loss of cellular RNA content (but not DNA or protein) and the rapid and complete inhibition of protein synthesis in tetrahydroaminoacridine-treated cells (lowest concentration tested was 0.5 mmol/L or 58 micrograms/10(6) cells). Thus tetrahydroaminoacridine treatment appears to aggregate hepatocellular nucleic acids, and in doing so adversely affects ribosomal function and protein synthesis. We propose that these adverse effects of exposure to tetrahydroaminoacridine are responsible for tetrahydroaminoacridine-induced hepatotoxicity.

Nitrofurantoin: mechanism of action and implications for resistance development in common uropathogens.

Nitrofurantoin is an effective urinary tract antibacterial to which no clinically significant resistance development has occurred. We have previously shown that nitrofurantoin susceptibility in bacteria correlates with the presence of bacterial nitroreductases which convert nitrofurantoin to highly reactive electrophilic intermediates. These intermediates were shown to attack bacterial ribosomal proteins non-specifically, causing complete inhibition of protein synthesis. In the present study, we confirm previous reports that low concentrations of nitrofurantoin specifically inhibit inducible enzyme synthesis in bacteria, and show that this inhibition occurs at levels equivalent to the MICs of nitrofurantoin for several bacterial species. Our previous studies had shown that nitrofurantoin at different concentrations interacts with bacterial ribosomal proteins in qualitatively the same fashion; we now report that quantitative differences are seen in the labelling observed at different nitrofurantoin concentrations and discuss these differences as they may relate to the inhibition of inducible enzyme synthesis. In addition, we have now demonstrated the existence of a novel mechanism of action for nitrofurantoin which does not require the production of reactive nitrofurantoin metabolites by bacterial reductases. The lack of clinically significant bacterial resistance development to nitrofurantoin is likely due to the combination of nitrofurantoin's multiple sites of attack and multiple mechanisms of action.

Patterns of protein synthesis in various cells after extreme heat shock

The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Azal and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 °C was carried out. Shock at 42 °C reduced the total synthetic rate of proteins in all three cell lines and induced “classical” heat-shock protein with a mass of 70 kDa (hsp70). Heat shock at 44 °C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 °C teratocarcinoma cells were able to accumulate anomalous amounts of hsp70 despite hsp70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.

Induction and superinduction of auxin-responsive mRNAs with auxin and protein synthesis inhibitors.

We have identified a class of small mRNAs (approximately 0.5 kilobases), referred to as small auxin-up RNAs (SAURs), that increases in abundance within minutes after auxin application to excised elongating hypocotyl sections of soybean. In this study, we present evidence that SAURs accumulate in the absence of auxin when protein synthesis is inhibited. Superinduction of SAURs occurs if the synthetic auxin 2,4-dichlorophenoxyacetic acid is added under conditions where protein synthesis is inhibited. Transcription run-on experiments with isolated nuclei show that, unlike 2,4-dichlorophenoxyacetic acid, protein synthesis inhibitors do not activate transcription of the SAUR genes. These results suggest that protein synthesis inhibitors act by stabilizing SAURs and that some labile protein(s) are involved in the rapid turnover of SAURs. This stabilization is not observed with GH3, another auxin-inducible mRNA. Whether induced with 2,4-dichlorophenoxyacetic acid or cycloheximide, SAURs are primarily expressed in epidermal and cortical cells of elongating hypocotyl sections, with little or no expression in vascular tissue. Unlike 2,4-dichlorophenoxyacetic acid-induced SAUR accumulation, the increase in abundance of SAURs induced by cycloheximide is transient, with a peak approximately 1 h after inhibitor addition. Complete inhibition of protein synthesis is not required for SAUR accumulation in the presence of protein synthesis inhibitors.

Stabilization of tubulin mRNA by inhibition of protein synthesis in sea urchin embryos.

An increased level of unpolymerized tubulin caused by depolymerization of microtubules in sea urchin larvae resulted in a rapid loss of tubulin mRNA, which was prevented by nearly complete inhibition of protein synthesis. Results of an RNA run-on assay indicated that inhibition of protein synthesis does not alter tubulin gene transcription. Analysis of the decay of tubulin mRNA in embryos in which RNA synthesis was inhibited by actinomycin D indicated that inhibition of protein synthesis prevents the destabilization of tubulin mRNA. The effect was similar whether mRNA was maintained on polysomes in the presence of emetine or anisomycin or displaced from the polysomes in the presence of puromycin or pactamycin; thus, the stabilization of tubulin mRNA is not dependent on the state of the polysomes after inhibition of protein synthesis. Even after tubulin mRNA declined to a low level after depolymerization of microtubules, it could be rescued by treatment of embryos with inhibitors of protein synthesis. Tubulin mRNA could be induced to accumulate prematurely in gastrulae but not in plutei if protein synthesis was inhibited, an observation that is indicative of the importance of the autogenous regulation of tubulin mRNA stability during embryogenesis. Possible explanations for the role of protein synthesis in the control of mRNA stability are discussed.